Acoustic radiation for ejecting and monitoring pathogenic fluids

ABSTRACT

Method and system for monitoring for a change in the amount and/or concentration of a pathogen in a pathogenic fluid. The method includes providing a pathogen-impermeable enclosure enclosing the pathogenic fluid, wherein the pathogenic fluid includes a pathogen and a carrier fluid. Additionally, the method includes acoustically monitoring for a change in the amount and/or concentration of the pathogen enclosed in the pathogen-impermeable enclosure. The acoustically monitoring for a change in the amount and/or concentration of the pathogen enclosed in the pathogen-impermeable enclosure includes generating acoustic radiation directed towards the pathogen-impermeable enclosure, transmitting the acoustic radiation through the pathogen-impermeable enclosure or reflecting the acoustic radiation by the pathogenic fluid, receiving the acoustic radiation transmitted through the pathogen-impermeable enclosure or receiving the acoustic radiation reflected by the pathogenic fluid, and analyzing the received acoustic radiation.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. patent application Ser. No.12/174,824, filed Jul. 17, 2008, abandoned, which is a divisional ofU.S. patent application Ser. No. 10/199,907, filed Jul. 18, 2002, nowU.S. Pat. No. 7,405,072, both applications being hereby incorporated byreference in their entirety.

TECHNICAL FIELD

The invention relates generally to the use of acoustic radiation inconjunction with pathogenic fluids. In particular, the invention relatesto the acoustic monitoring of the pathogenic contents within areservoir, as well as to the acoustic ejection of pathogenic fluiddroplets. The invention also relates to the use of acoustic radiation inconjunction with pathogen-impermeable enclosures.

BACKGROUND

Cultures containing cellular matter may be employed to study pathogenicmaterial such as bacteria and viruses. For example, pathogen-impermeablecontainers having an interior surface coated with a layer of solid orsemisolid medium within which cells are grown may be inoculated with thedesired type of cells. After the cells are subjected to conditionsappropriate for cultivation, they may be removed from the containers asa suspension and may optionally be concentrated. Also, if desired, viralmatter may be extracted from the cells after removal from thecontainers.

Pathogenic substances, however, including viruses (such as the humanimmunodeficiency virus (HIV), rabies, and herpes) and bacteria (such asbacillus anthracis, yersinia pestis, and those of the streptococcusgenus), must be handled with extreme care to prevent release of thepathogen. In addition, there exists a need in pharmaceutical,biotechnological, and other scientific industries to quickly screen,identify, and/or process large numbers or varieties of fluids,pathogenic or otherwise. As a result, much attention has been focused ondeveloping efficient, precise, and accurate fluid handling methods thatmay be used, for example, to carry out screening assays and/orcombinatorial techniques. Since fluids used in pharmaceutical,biotechnological, and other scientific industries may be rare and/orexpensive, techniques capable of handling small volumes of fluidsprovide readily apparent advantages over those requiring relativelylarger volumes. Furthermore, as pathogenic fluids represent a potentialsafety hazard, it is also desirable to reduce the quantities used tocarry out studies or investigations involving such substances.

Typically, fluids for use in combinatorial methods are provided as acollection or library of organic and/or biological compounds. In manyinstances, such libraries and collections are provided in a well plateformat for screening and/or processing. Well plates are typicallysingle-piece in construction and comprise a plurality of identicalwells, wherein each well is adapted to contain a small volume of fluid.Such well plates are commercially available in standardized formats andsizes, and may contain, for example, 96, 384, 1536, or 3456 wells perwell plate. Fluids are typically transferred from such well plates,e.g., during formatting and reformatting procedures, using devices thatrequire contact between the fluid to be transferred and a solid surfaceof a device. For example, capillaries (Eppendorf-type or otherwise)having small interior channels are commonly employed for sample fluidhandling by submerging their ends into a pool of sample. Pipettingsystems, whether automated, robotic, or otherwise, that have submergibletips may be employed as well. Contact between the solid surface and thefluid to be transferred typically results in surface wetting thatrepresents a source of unavoidable fluid waste as well as a source ofpotential pathogenic contamination. In addition, if more than one fluidis to contact an interior or exterior solid surface of a non-disposablecapillary or pipette tip, the surface must be washed between sampletransfers in order to eliminate cross contamination and samplecarry-over. The liquid biohazard waste created from this wash processmust then be disposed of and rendered harmless. It would be desirable toavoid liquid waste generation from repeated wash processes and eliminateadditional storage and disposal costs. Disposable pipette tips orcapillaries may be used to avoid the generation of liquid waste.However, disposal of solid waste also incurs storage and disposal costs.

Thus, there is a need for fluid handling systems that enable safe andconvenient handling, formatting, and reformatting of potentiallydangerous bacterial, viral, and other pathogenic specimens. Such fluidhandling systems may be used, for example, to perform clinicaldiagnostic tests, engage in high-throughput drug screening, and carryout growth inhibition studies. In order to ensure that pathogens are notreleased during fluid handling procedures, pathogen-impermeableenclosures such as glove boxes may be used to contain the pathogenicspecimens. Small volume pathogenic cultures, however, often requirecomplicated manual manipulations, which are not easily carried out usingglove boxes; thus, performing such procedures in a glove box wouldlikely introduce error during handling and result in possibleunwarranted experimental conclusions. Various automated devices tocontrol fluid transfer in closed systems for culturing living pathogenshave been developed. U.S. Pat. No. 6,022,742 to Kopf, for example,describes one such automated device.

The use of acoustic energy in printing technology is also known. Forexample, U.S. Pat. No. 4,308,547 to Lovelady et al. describes a liquiddrop emitter that utilizes acoustic principles to eject liquid from abody of liquid onto a moving document in order to form characters orbarcodes thereon. Lovelady et al. is directed to a nozzleless inkjetprinting apparatus, wherein controlled drops of ink are propelled by anacoustical force produced by a curved transducer at or below the surfaceof the ink. In contrast to capillaries, syringes, pipettes, inkjet printheads, and other such fluid dispensing devices that employ a nozzle,tip, or tubing for fluid transfer, nozzleless fluid ejection devices asdescribed in the aforementioned patent do not contain componentsrequiring cleaning and/or disposal after use. In addition, disadvantagesassociated with nozzles or tips in fluid dispensing systems, includingclogging, misdirected fluid, improperly sized droplet formation, and thelike, are avoided. More recently, acoustic ejection has been employed incontexts other than in ink printing applications. For example, U.S.Patent Application Publication No. 20020037579 to Ellson et al.describes the use of focused acoustic radiation to dispense fluids withsufficient accuracy and precision to prepare biomolecular arrays from aplurality of reservoirs.

Acoustic radiation has also been used to assess the contents of acontainer adapted to contain a liquid. Traditionally, the contents maybe assessed by contacting a sensor with the liquid (see U.S. Pat. No.5,507,178 to Dam), or by transmitting acoustic radiation through an opentop of a container and detecting radiation reflected from an air-liquidinterface of the container back to the sensor (see U.S. Pat. No.5,880,364 to Dam). More recently, U.S. patent application Ser. No.10/010,972, Publication No. 20030101819, “Acoustic Assessment of Fluidsin a Plurality of Reservoirs,” inventors Mutz, Ellson, and Foote, filedon Dec. 4, 2001, describes an improved acoustic assessment techniquethat involves the transmission of acoustic radiation through a reservoirto assess the fluid contents within the reservoir without requiringdirect contact with the fluid contents therein. By analyzing acharacteristic of the acoustic radiation transmitted through the fluid,various properties of the fluid within the reservoir may be determined.This type of acoustic monitoring may be used advantageously inconjunction with optically opaque reservoirs.

Similarly, focused acoustic energy recently has been used inapplications involving biological matter such as living cells. Forexample, a number of U.S. patent applications describe the use offocused acoustic radiation to manipulate and sort cells. See U.S. PatentApplication Publication No. 20020064808 to Mutz et al.; U.S. patentapplication Ser. No. 09/999,166, Publication No. 20020142286, filed Nov.29, 2001, for “Focused Acoustic Energy for Ejection Cells from a Fluid,”inventors Mutz and Ellson, assigned to Picoliter, Inc. (Mountain View,Calif.); U.S. Patent Application Publication No. 20020064809 to Mutz etal.; and U.S. patent application Ser. No. 10/040,926, Publication No.20020090720, filed Dec. 28, 2001, for “Focused Acoustic Ejection CellSorting System and Method,” inventors Mutz, Ellson, and Lee, assigned toPicoliter, Inc. (Mountain View Calif.). Furthermore, the use of focusedacoustic radiation has been described for preparing and analyzing acellular sample surface. (See U.S. patent application Ser. No.10/087,372, Publication No. 20020171037, filed Mar. 1, 2002, entitled“Method and System Using Acoustic Ejection for Preparing and Analyzing aCellular Sample Surface,” inventors Ellson, Mutz, and Caprioli.)

The use of focused acoustic energy in the context of applicationsinvolving pathogenic fluids, however, has previously been unknown in theart. Thus, through the use of focused acoustic radiation, the inventionprovides previously unrealized opportunities in pathogenic studies.

SUMMARY OF THE INVENTION

In a first embodiment, the invention relates to a method for dispensingone or more droplets of a fluid containing a pathogen. The methodinvolves providing the pathogen-containing fluid in a reservoir andapplying focused radiation to the pathogen-containing fluid in thereservoir in a manner effective to eject a droplet of the fluidtherefrom. Typically, focused acoustic radiation is employed to carryout the invention. In addition, the invention may be used in conjunctionwith any of a number of different types of pathogens. The pathogen maybe a toxin, virus, and/or bacterium.

In some instances, the pathogen-containing fluid may be comprised of acarrier fluid and a plurality of discrete pathogenic particles. Inaddition, a plurality of discrete nonpathogenic particles may be presentin the carrier fluid as well. Accordingly, the inventive method mayinvolve locating a discrete pathogenic particle in the carrier fluidusing focused radiation. When focused acoustic radiation is employed,the location of the pathogenic particle may be detected by virtue of oneor more acoustic properties, such as acoustic impedance, which ensuresthat the ejected droplet contains the pathogenic particle. In somecases, however, the invention may be used to eject droplets containingnonpathogenic particles or no particles at all.

Thus, the invention also provides a method for selecting a localizedvolume in a pathogenic fluid for removal from a reservoir. When apathogen-containing fluid is provided in a reservoir and is comprised ofa plurality of particles and a carrier fluid, the localized volume maybe acoustically located and optionally removed. The localized volume maycontain zero, one, or more particles. Furthermore, the localized volumemay or may not be pathogenic. In other words, the invention may be usedto sort pathogenic from nonpathogenic fluids and particles and viceversa.

The invention may be used to deposit a droplet of fluid on a designatedsite of a substrate surface, typically by positioning the substrate sothat the designated site is in droplet-receiving relationship withrespect to the reservoir. In some instances, additional droplets offluid are deposited on the substrate surface from the same reservoir, orfrom different reservoirs. When the invention provides a plurality ofreservoirs, each reservoir typically contains a different fluid. In anycase, droplets may be deposited on the substrate surface at the samesite or at different designated sites. When the droplets are depositedat different designated sites, the sites may form an array of sites.

Thus, in some instances, the method may be used to determine whether thepathogen-containing fluid droplet interacts with a compound of interest.This may be carried out by either ensuring that a compound of interestis present at the designated site prior to the deposition of thedroplet, or by depositing a compound of interest at the designated siteafter the deposition of the droplet. The compound of interest, forexample, may be deposited on the designated site using focused radiationas well.

Once deposited, a droplet on the substrate surface may be isolated in apathogen-impermeable enclosure. When a plurality of droplets isdeposited, the droplets may be either individually isolated in aplurality of enclosures, or collectively isolated in the same enclosure.In either case, the pathogen-impermeable enclosure or enclosures may beformed by placing a pathogen-impermeable cover in sealing contact withthe substrate.

In another embodiment, the invention relates to a method for sealing afluid in a pathogen-impermeable enclosure. The method involves firstproviding the fluid in a reservoir and positioning a substrate so that adesignated site on a surface thereof is in droplet-receivingrelationship with respect to the reservoir. Then, focused radiation isapplied to the fluid in the reservoir in a manner effective to eject adroplet of the fluid therefrom onto the substrate surface at thedesignated site. The fluid droplet at the designated site is then sealedin the pathogen-impermeable enclosure. As before, this method typicallyemploys focused acoustic radiation.

The enclosure is typically sealed after introducing a pathogenic fluidtherein, to ensure that the pathogen is not released. The pathogenicfluid droplet may be ejected from the reservoir or dispensed fromelsewhere. Thus, the pathogenic fluid droplet may be deposited at thedesignated site before, during, or after focused radiation is applied tothe reservoir to eject a droplet of fluid therefrom. In some instances,however, the enclosure is sealed to ensure that no pathogen isintroduced therein. That is, the pathogen may be controllably sealed inor sealed out of the enclosure. In either case, the pathogen-impermeableenclosure is typically opened so as to expose the designated site withinthe enclosure to the reservoir, and sealed after a fluid droplet hasbeen placed in the enclosure. In some instances, thepathogen-impermeable enclosure may be formed from a cover and thesubstrate. In such a case, sealing the enclosure may involve placing thecover and the substrate in sealing contact with each other.

Often, droplets from the reservoir are deposited on the substratesurface. In some instances, however, a plurality of reservoirs eachcontaining a different fluid is provided and a droplet from eachreservoir is deposited on the substrate surface. In some instances,droplets are deposited at the same designated site. In other instances,the droplets are deposited at different designated sites. The differentdesignated sites may form an array of sites. When fluid droplets aredeposited at different designated sites, the droplets may all be sealedin a pathogen-impermeable enclosure, either together in the samecompartment, or isolated in separate compartments of thepathogen-impermeable enclosure.

In a further embodiment, the invention provides a device for dispensingone or more droplets of fluid. The device includes a reservoir adaptedto contain a fluid, an ejector for applying focused radiation to thereservoir in a manner effective to eject a droplet of fluid from thereservoir, a means for positioning a substrate to receive a droplet offluid from the reservoir, and a pathogen-impermeable enclosure forisolating the reservoir and substrate therein. Typically, the ejector isan acoustic ejector. Although ordinary inkjet technologies may beemployed, it is preferred that the ejector is a nozzleless acousticdevice that employs an acoustic generator and a focusing means forfocusing the acoustic radiation generated thereby.

The inventive device may include additional features that serve toenhance the performance of the device. For example, the device mayfurther include a means for manually manipulating items within theenclosure without compromising the pathogenic impermeability of theenclosure. In addition, a locating means may be provided for locating adiscrete particle in the pathogenic fluid. When the device includes anacoustic generator, the locating means may include an analyzer foranalyzing acoustic radiation generated by the acoustic generator. Suchan analyzer is typically positioned to receive acoustic radiationgenerated by the acoustic generator and transmitted through fluidcontained in the reservoir. In some instances, the analyzer ispositioned to receive acoustic radiation reflected by a free fluidsurface contained in the reservoir. In such a case, the analyzer mayinclude a component common to the acoustic generator, e.g., apiezoelectric element.

Typically, the reservoir is detachable from the device and may beadapted for single use. In addition, the device may further include apathogen-impermeable cover. Such a cover may be adapted to make sealingcontact with the reservoir in order to contain a pathogenic fluidtherein.

Optionally, the device includes a plurality of reservoirs. In someinstances, the reservoirs are provided in a single-piece unit, such aswhen the reservoirs represent wells of a well plate. The reservoirs arepreferably substantially acoustically indistinguishable from each other.In addition, the device may further include a means for successivelypositioning the acoustic device in an acoustically coupled relationshipwith each of the reservoirs.

In still another embodiment, the invention relates to a method formonitoring a change in the amount and/or concentration of a pathogen ina pathogenic fluid. The method involves providing a pathogen-impermeableenclosure that encloses a pathogenic fluid comprising a pathogen and acarrier fluid, and acoustically monitoring for a change in the amountand/or concentration of the pathogen enclosed in thepathogen-impermeable enclosure. The method may be used to measure eitheran increase or a decrease in pathogen content. Thus, the method isparticularly suited for carrying out processes and/or assays in whichpathogen content and/or concentration is altered. For example,additional material, e.g., nutrients in a culturing solution, may beintroduced into the enclosure, which then may be subjected to atemperature change. The temperature may be selected to facilitate anincrease or decrease in the amount and/or concentration of the pathogenin the enclosure.

In a further embodiment, the invention relates to a method for detectingfor an interaction between a fluid and a compound. The method involves:(a) providing a reservoir containing the fluid; (b) depositing thecompound onto a designated site on a surface of a substrate; (c)positioning the substrate so that the designated site is indroplet-receiving relationship with respect to the reservoir; (d)applying focused radiation to the fluid in the reservoir in a mannereffective to eject a droplet of the fluid therefrom onto the substratesurface at the designated site; (e) sealing the fluid droplet and thecompound at the designated site in a pathogen-impermeable enclosure; and(f) detecting for an interaction between the fluid and the compound.Either the compound, fluid, or both may be pathogenic. The interactionmay be detected through various means such as acoustic, optic,fluorescence, magnetic and/or electrical means.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1D, collectively referred to as FIG. 1, schematicallyillustrate in simplified cross-sectional view the operation of anenclosed system that uses focused acoustic radiation to study theinteraction between a candidate compound and a bacterial pathogen. FIG.1A shows the use of acoustic radiation to locate a pathogenic bacterialparticle near the surface of a fluid in a reservoir. FIG. 1B shows theejection of a droplet containing a bacterial pathogen from the reservoironto a designated site of a substrate surface. FIG. 1C illustrates theplacement of the substrate in acoustically coupled relationship with anacoustic analyzer and an initial acoustic assessment of the pathogeniccontents of the well. FIG. 1D illustrates a subsequent assessment of thepathogenic contents of the well after exposure to culturing conditions.

FIG. 2 schematically illustrates in simplified cross-sectional view theacoustic assessment of the pathogenic contents of pathogen-impermeableenclosure in transmissive mode.

DETAILED DESCRIPTION OF THE INVENTION

Before describing the present invention in detail, it is to beunderstood that, unless otherwise indicated, this invention is notlimited to specific fluids, acoustic devices, substrates, or the like,as such may vary. It is also to be understood that the terminology usedherein is for the purpose of describing particular embodiments only, andis not intended to be limiting.

It must be noted that, as used in this specification and the appendedclaims, the singular forms “a,” “an,” and “the” include plural referentsunless the context clearly dictates otherwise. Thus, for example, theterm “a fluid” is intended to mean a single fluid or a mixture offluids, “a reservoir” is intended to mean one or more reservoirs, “apathogen” refers to a single pathogen as well as a plurality ofpathogens.

In describing and claiming the present invention, the followingterminology will be used in accordance with the definitions set outbelow.

The terms “acoustic coupling” and “acoustically coupled” as used hereinrefer to a state wherein an object is placed in direct or indirectcontact with another object so as to allow acoustic radiation to betransferred between the objects without substantial loss of acousticenergy. When two entities are indirectly acoustically coupled, an“acoustic coupling medium” is needed to provide an intermediary throughwhich acoustic radiation may be transmitted. Thus, an acoustic devicemay be acoustically coupled to a fluid, such as by immersing theacoustic device in the fluid, or by interposing an acoustic couplingmedium between the acoustic device and the fluid, in order to transferacoustic radiation generated by the acoustic device through the acousticcoupling medium and into the fluid.

The terms “acoustic radiation” and “acoustic energy” are usedinterchangeably herein and refer to the emission and propagation ofenergy in the form of sound waves. As with other waveforms, acousticradiation may be focused using a focusing means, as discussed below.

The term “array” as used herein refers to a two-dimensional arrangementof features, such as an arrangement of reservoirs (e.g., wells in a wellplate) or an arrangement of different moieties, including ionic,metallic, or covalent crystalline (e.g., molecular crystalline),composite, ceramic, vitreous, amorphous, fluidic, or molecular materialson a substrate surface (as in an oligonucleotide or peptidic array).Arrays are generally comprised of regular features ordered in, forexample, a rectilinear grid, parallel stripes, spirals, and the like,but nonordered arrays may be advantageously used as well. An array isdistinguished from the more general term “pattern” in that patterns donot necessarily contain regular and ordered features.

The terms “biomolecule” and “biological molecule” are usedinterchangeably herein to refer to any organic molecule that is, was, orcan be a part of a living organism, regardless of whether the moleculeis naturally occurring, recombinantly produced, or chemicallysynthesized in whole or in part. The terms encompass, for example,monomeric molecules, such as nucleotides, amino acids, andmonosaccharides, oligomeric and polymeric species, such asoligonucleotides and polynucleotides, peptidic molecules, such asoligopeptides, polypeptides, and proteins, saccharides, such asdisaccharides, oligosaccharides, polysaccharides, mucopolysaccharides,and peptidoglycans (peptido-polysaccharides), and the like. The termsalso encompass ribosomes, enzyme cofactors, pharmacologically activeagents, and the like. Additional information relating to the term“biomolecule” can be found in U.S. Patent Application Publication No.20020037579 by Ellson et al.

The teem “enclosure” is used herein in its ordinary sense and refers toanything that encloses. Examples of enclosures include, but are notlimited to, bottles, boxes, canisters, cans, cartons, cartridges,containers, drums, jars, and vials.

The term “fluid” as used herein refers to matter that is nonsolid, or atleast partially gaseous and/or liquid, but not entirely gaseous. A fluidmay contain a solid that is minimally, partially, or fully solvated,dispersed, or suspended. Examples of fluids include, without limitation,aqueous liquids (including water per se and salt water) and nonaqueousliquids such as organic solvents and the like. As used herein, the term“fluid” is not synonymous with the term “ink,” in that ink must containa colorant and may not be gaseous. Thus, the term “bodily fluid” as usedherein refers to any fluid that can be extracted from an individual'sbody, pathogenic or nonpathogenic. When the individual is a mammal,e.g., human, the term includes fluids such as blood, plasma, serum,interstitial fluid, lymph, bile, spinal fluid, amnionic fluid, urine,saliva, vaginal fluid, and etc.

The term “focusing means” refers to a means for causing waves toconverge at a focal point. When acoustic radiation is involved, an“acoustic focusing means” causes acoustic radiation to converge at afocal point either by a device separate from the acoustic energy sourcethat acts like an optical lens, or by the spatial arrangement ofacoustic energy sources to effect convergence of acoustic energy at afocal point by constructive and destructive interference. An acousticfocusing means may be as simple as a solid member having a curvedsurface, or it may include complex structures such as those found inFresnel lenses, which employ diffraction in order to direct acousticradiation. Suitable focusing means also include phased array methods asare known in the art and described, for example, in U.S. Pat. No.5,798,779 to Nakayasu et al. and Amemiya et al. (1997) Proceedings ofthe 1997 IS&T NIP13 International Conference on Digital PrintingTechnologies, pp. 698-702.

The term “impermeable” is used in its ordinary sense to mean notpermitting something to pass through. Similarly, the term “permeable” isused herein in its ordinary sense and means “not impermeable.”Typically, the term “impermeable” is used to describe certainenclosures, and the term “permeable” is used to describe certain“substrates” or “surfaces.” Thus, a “pathogen-impermeable enclosure”refers to an enclosure that does not allow a pathogen to pass through,and a “permeable substrate” and a “substrate having a permeable surface”refer to a substrate or surface, respectively, that can be permeatedwith water or other fluid.

The terms “library” and “combinatorial library” are used interchangeablyherein to refer to a plurality of chemical or biological moietiesarranged in a pattern or an array such that the moieties areindividually addressable. In some instances, the plurality of chemicalor biological moieties is present on the surface of a substrate, and inother instances, the plurality of moieties represents the contents of aplurality of reservoirs. Preferably, but not necessarily, each moiety isdifferent from each of the other moieties. The moieties may be, forexample, peptidic molecules and/or oligonucleotides.

The term “moiety” refers to any particular composition of matter, e.g.,a molecular fragment, an intact molecule (including a monomericmolecule, an oligomeric molecule, and a polymer), or a mixture ofmaterials (for example, an alloy or a laminate).

“Optional” or “optionally” means that the subsequently describedcircumstance may or may not occur, so that the description includesinstances where the circumstance occurs and instances where it does not.

The terms “pathogen” and “pathogenic” as used herein refer to any agentthat is capable of causing disease and/or a toxic response in anindividual. The individual may be a human, an animal (mammalian orotherwise), or on occasion, a plant. Typically, a pathogen referred toherein is a bacterium or virus, but may also be an organic toxin such asstrychnine or botulinum, or an inorganic toxin such as arsenic or sodiumcyanide. Often, pathogens are biomolecular in nature. Thus, exemplarybacterial pathogens include, but are not limited to, bacteria of thefollowing genera, Campylobactera, Bacteroides, Bordetella, Haemophilus,Pasteurella, Francisella, Actinobacillus, Klebisella, Moraxella,Pseudomonas, pneumococci, Proteus, Ornithobacterium, Staphylococci andStreptococci. Salmonella is another exemplary genus of pathogenicbacteria and includes species such as Salmonella typhimurium, Salmonellaenteriditis, Salmonella gallinarum, Salmonella pullorum, Salmonellaarizona, Salmonella heidelberg, Salmonella anatum, Salmonella hadar,Salmonella agana, Salmonella montevideo, Salmonella kentucky, Salmonellainfantis, Salmonella schwarzengrund, Salmonella saintpaul, Salmonellabrandenburg, Salmonella instanbul, Salmonella cubana, Salmonellabredeney, Salmonella braenderup, Salmonella livingstone, Salmonellaberta, Salmonella california, Salmonella senfenberg, and Salmonellambandaka. Mycobacterium is another type of pathogenic bacteria that isparticularly harmful to humans and includes species such asMycobacterium tuberculosis, Mycobacterium avium, Mycobacteriumparatuberculosis, Mycobacterium bovis and Mycobacterium leprae.

Anaerobic bacterial pathogens include, for example, those in the generaPeptostreptococci, Actinomyces, Clostridium, Anaerobiospirillum,Fusobacterium, and Bilophila. Thus, exemplary anaerobic bacterialpathogens include, for example, Peptostreptococci asaccharolyticus,Peptostreptococci magnus, Peptostreptococci micros, Peptostreptococciprevotii, Porphyromonas asaccharolytica, a Porphyromonas canoris,Porphyromonas gingivalis, Porphyromonas macaccae, Actinomyces israelii,Actinomyces odontolyticus, Clostridium innocuum, Clostridiumclostridioforme, Clostridium difficile, Bacteroides tectum, Bacteroidesureolyticus, Bacteroides gracilis (Campylobacter gracilis), Prevotellaintermedia, Prevotella heparinolytica, Prevotella oris-buccae,Prevotella bivia, Prevotella melaninogenica, Fusobacterium naviforme,Fusobacterium necrophorum, Fusobacterium varium, Fusobacterium ulcerans,Fusobacterium russii, and Bilophila wadsworthia.

Exemplary upper respiratory pathogenic bacteria include, for example,those in the genera Pseudomonas and Legionella. Thus exemplary upperrespiratory upper respiratory pathogens include, Pseudomonas aeruginosa,Legionella dumoffii, Legionella longbeacheae, Legionella micdadei,Legionella oakridgensis, Legionella feelei, Legionella anisa, Legionellasainthelensi, Legionella bozemanii, Legionella gormanii, Legionellawadsworthii, and Legionella jordanis.

Nonbacterial pathogens include, but are not limited to viruses and fungiand prions. Exemplary viral pathogens include, generally, those ofclasses I-VI, and more specifically, hepatitis viruses types A-E, ebolaviruses, human papilloma viruses, keratoconjunctivitis viruses,Parvoviruses, erythroviruses, dependoviruses, echo viruses,enteroviruses, Epstein-Barr viruses, equine arteritis virus, equinecoital exanthema virus, equine encephalosis virus, feline sarcomaviruses, hantaviruses, herpes viruses, human inmmunodeficiency viruses,human T-cell leukaemia viruses, influenza viruses types A-C, JC viruses,Kirsten sarcoma viruses, Lassa viruses, Machupo viruses, Marburgviruses, mastadenoviruses, measles virus, Mengo viruses, Moloney murineleukemia viruses, Newcastle Disease virus, orbiviruses, polio viruses,retroviruses, simian immunodeficiency viruses, small pox viruses,Tamiami viruses, and tobacco mosaic viruses. Fungal pathogens include,for example, Pyrenophora tritici-repentis, Drechslera sorokiniana,Rhizoctonia cerealis, Fusarium graminearum, Fusarium culmorum,Microdochium nivale, Pseudocercosporella herpotrichoides,Pseudocercosporella herpotrichoides, Septoria nodorum, Septoria tritici,Cladosporium herbarum, Cercospora arachidicola, Helminthosporiumsativum, Pyrenophora teres, and Pyrenophora tritici-repentis. It shouldbe noted that these pathogens are enumerated in no particular order andsome overlap may occur. Other pathogens are known in the art andidentified, for example, in Sherris Medical Microbiology: AnIntroduction to Infectious Diseases, 3rd Ed. (Appleton & Lange,Stamford, Conn., 1994).

Thus, the term “pathogen-containing fluid” refers to nonsolid matterthat is completely or partially pathogenic in nature. Such a fluid, forexample, may be comprised of liquid that contains a pathogen minimally,partially, or fully solvated, dispersed, or suspended therein. Examplesof pathogen-containing fluids include, without limitation, a culturingmedium containing bacterial or viral infectious agents.

Similarly, the “nonpathogenic” refers to matter that is not pathogenic,i.e., any agent that is not likely to cause disease or a toxic response.Nonpathogenic particles, for example, include, without limitation,beneficial cellular matter such as lactobacilli, yeast, epidermal cells,beads and the like. Nonpathogenic fluids include, for example, sterilesaline, glucose solutions, and the like.

The term “radiation” is used in its ordinary sense and refers toemission and propagation of energy in the form of a waveform disturbancetraveling through a medium such that energy is transferred from oneparticle of the medium to another without causing any permanentdisplacement of the medium itself. Thus, radiation may refer, forexample, to electromagnetic waveforms as well as acoustic vibrations.

The term “reservoir” as used herein refers to a receptacle or chamberfor containing a fluid. In some instances, a fluid contained in areservoir necessarily will have a free surface, e.g., a surface thatallows acoustic radiation to be reflected therefrom or a surface fromwhich a droplet may be acoustically ejected. A reservoir may also be alocus on a substrate surface within which a fluid is constrained.

The term “substrate” as used herein refers to any item having a surfaceonto which one or more fluids may be deposited. The substrate may beconstructed in any of a number of forms including, for example, wafers,slides, well plates, or membranes. In addition, the substrate may beporous or nonporous as required for deposition of a particular fluid.Suitable substrate materials include, but are not limited to, supportsthat are typically used for solid phase chemical synthesis, such aspolymeric materials (e.g., polystyrene, polyvinyl acetate, polyvinylchloride, polyvinyl pyrrolidone, polyacrylonitrile, polyacrylamide,polymethyl methacrylate, polytetrafluoroethylene, polyethylene,polypropylene, polyvinylidene fluoride, polycarbonate, anddivinylbenzene styrene-based polymers), agarose (e.g., SEPHAROSE®),dextran (e.g., SEPHADEX®), cellulosic polymers and otherpolysaccharides, silica and silica-based materials, glass (particularlycontrolled pore glass, or “CPG”), functionalized glasses, and ceramics,as well as such substrates treated with coatings that cover the entiretyor a portion of a surface, e.g., treated with microporous polymers(particularly cellulosic polymers such as nitrocellulose), microporousmetallic compounds (particularly microporous aluminum), antibody-bindingproteins (available from Pierce Chemical Co., Rockford, Ill.), bisphenolA polycarbonate, poly-L-lysine and the like. Such coatings may bedeposited via acoustic ejection or other means, to form arrays or otherpatterns on the substrate surface. Additional information relating tothe term “substrate” can be found in U.S. Patent Application PublicationNo. 20020037579 to Ellson et al.

The invention thus generally relates to methods that employ focusedradiation to eject droplets of a fluid from a reservoir and to handlepathogenic materials. Typically, focused acoustic radiation is employedto eject droplets of a pathogenic fluid from a reservoir. In addition,the inventive method may be used in conjunction with apathogen-impermeable enclosure. For example, the method may involvepositioning a substrate so that a designated site on a substrate surfaceis placed in droplet-receiving relationship with respect to thereservoir. After focused radiation is applied to the fluid in thereservoir such that a droplet of the fluid is deposited at thedesignated site, the droplet may be sealed in the pathogen-impermeableenclosure.

The invention also provides a method of monitoring for a change in theamount and/or concentration of a pathogen in a pathogenic fluid. Insteadof using focused acoustic radiation to eject droplets from a reservoir,a pathogen-impermeable enclosure is provided enclosing a pathogenicfluid that is comprised of a pathogen and a carrier fluid. Acousticradiation is then generated to monitor for a change in the amount and/orconcentration of the pathogen enclosed in the pathogen-impermeableenclosure. Generally, the invention is suited for use with biologicalpathogens such as viral and bacterial matter.

FIG. 1 illustrates in simplified cross-sectional view a system forstudying the interaction between a candidate compound and a bacterialpathogen. As with all figures referenced herein, in which like parts arereferenced by like numerals, FIG. 1 is not to scale, and certaindimensions may be exaggerated for clarity of presentation. The system 1includes an enclosure 11 that serves to enclose the other components ofthe system. A reservoir 13 is provided containing a pathogen-containingfluid 15 having a fluid surface 17. The pathogen-containing fluid iscomprised of at least one discrete bacterial pathogenic particle 19suspended in a carrier fluid 21. A plurality of pathogenic particles 19is depicted in FIG. 1A, though the pathogen-containing fluid may becomprised of a single pathogenic particle as well. As shown, reservoir13 may be axially symmetric, having a vertical wall 23 extending upwardfrom a circular reservoir base 25 and terminating at an opening 27. Thematerial and thickness of the reservoir base should be such thatacoustic radiation may be transmitted therethrough and into thepathogen-containing fluid 15 within the reservoir 13.

The system 1 also includes an acoustic device 29 comprised of anacoustic generator 31 for generating acoustic radiation and a focusingmeans 33 for focusing the acoustic radiation at a focal point within thefluid from which a droplet is to be ejected near the fluid surface. Theacoustic device may be used as an ejector and/or as an analyzer. Theacoustic generator contains a transducer, e.g., a piezoelectric element35, commonly shared by an analyzer. As shown, a combination unit 37 isprovided that both serves as a controller and a component of ananalyzer. Operating as a controller, the combination unit 37 providesthe piezoelectric element 35 with electrical energy that is convertedinto mechanical and acoustic energy. Operating as a component of ananalyzer, the combination unit receives and analyzes electrical signalsfrom the transducer. The electrical signals are produced as a result ofthe absorption and conversion of mechanical and acoustic energy by thetransducer 35.

As shown in FIG. 1, the focusing means 33 may comprise a single solidpiece having a concave surface 39 for focusing acoustic radiation, butthe focusing means may be constructed in other ways as discussed below.The acoustic device 29 is thus adapted to generate and focus acousticradiation near a fluid surface 17 when acoustically coupled to areservoir 13. The acoustic generator 31 and the focusing means 33 mayfunction as a single unit controlled by a single controller, or they maybe independently controlled, depending on the desired performance of thedevice.

There are a number of ways to acoustically couple the acoustic device 29to the reservoir 13 and thus to the fluid therein. One such approach isthrough direct contact as is described, for example, in U.S. Pat. No.4,308,547 to Lovelady et al., wherein a focusing means constructed froma hemispherical crystal having segmented electrodes is submerged in aliquid to be ejected. However, a preferred approach is to acousticallycouple the acoustic device to the reservoir 13 and fluid 15 withoutcontacting any portion of the acoustic device, e.g., the focusing means,with the fluid 15. To this end, as illustrated in FIG. 1, the acousticdevice is positioned in controlled and repeatable acoustic coupling withfluid 15 in reservoir 13. This typically involves direct or indirectcontact between the acoustic device and the external surface of thereservoir 13. When direct contact is used in order to acousticallycouple the acoustic device to the reservoir, it is preferred that thedirect contact be wholly conformal to ensure efficient acoustic energytransfer. That is, the acoustic device and the reservoir should havecorresponding surfaces adapted for mating contact. Thus, if acousticcoupling is achieved between the acoustic device and reservoir throughthe focusing means, it is desirable for the reservoir to have an outsidesurface that corresponds to the surface profile of the focusing means.Without conformal contact, efficiency and accuracy of acoustic energytransfer may be compromised. In addition, since many focusing means havea curved surface, the direct contact approach may necessitate the use ofa reservoir having a specially formed inverse surface.

Optimally, acoustic coupling is achieved between the acoustic device andthe reservoir through indirect contact, as illustrated in FIG. 1A. Inthis figure, an acoustic coupling medium 41 is placed between thefocusing means 33 and the base 25 of reservoir 13, with the acousticdevice and reservoir located at a predetermined distance from eachother. The acoustic coupling medium may be an acoustic coupling fluid,preferably an acoustically homogeneous material, in conformal contactwith both the acoustic focusing means 33 and each reservoir. Inaddition, it is important to ensure that the acoustic coupling medium issubstantially free of material having different acoustic properties thanthe fluid medium itself. Furthermore, it is preferred that the acousticcoupling medium be comprised of a material having acoustic propertiesthat facilitate the transmission of acoustic radiation withoutsignificant attenuation of acoustic pressure and intensity. Also, theacoustic impedance of the coupling medium should facilitate the transferof energy from the coupling medium into the container. As shown, thefirst reservoir 13 is acoustically coupled to the acoustic focusingmeans 33, such that an acoustic wave is generated by the acousticgenerator and directed by the focusing means 37 into the acousticcoupling medium 41, which then transmits the acoustic radiation into thereservoir 13.

Also provided is a substrate 51 having designated site 53 on a surface55 thereof. As shown, the substrate 51 may be a well that together witha cover 57 form an enclosure adapted to contain a volume of fluid, andthe designated site 53 may be located within the interior of theenclosure. A culturing fluid 59 for sustaining and growing bacterialpathogens and, optionally, for containing a candidate compound, may beprovided in the interior of the enclosure.

In operation as an analyzer, the acoustic device 33 may first bepositioned below reservoir 13, so as to be acoustically coupled to thereservoir through acoustic coupling medium 41. Once the acoustic deviceand the reservoir are in proper alignment, the transducer 35 of theacoustic generator 31, as depicted in FIG. 1A, is activated to produceacoustic radiation that is directed toward surface 17 of the reservoir,with the amount of energy being insufficient to eject fluid. This istypically accomplished by using an extremely short pulse (on the orderof tens of nanoseconds) relative to that required for droplet ejection(on the order of microseconds). The acoustic radiation will then travelin a generally upward direction toward the free fluid surface 17. Theacoustic radiation will be reflected under different circumstances.Typically, reflection will occur when there is a change in an acousticproperty of the medium through which the acoustic radiation istransmitted. Thus, this first emission of focused acoustic energypermits sonic detection of the presence of a bacterial pathogenicparticle sufficiently close to the surface for ejection by virtue ofreflection of acoustic energy by the particle. Methods for determiningthe position of the particles by sonic detection are readily apprehendedby those of ordinary skill in the art of acoustic microscopy and relatedarts. After a pathogenic particle is detected and located, otherproperties and/or characteristics of the carrier fluid and thepathogenic particle may be measured before the decision to eject is madeas described in U.S. patent application Ser. No. 10/010,972, PublicationNo. 20030101819, entitled “Acoustic Assessment of Fluids in a Pluralityof Reservoirs,” filed Dec. 4, 2001, by inventors Mutz, Ellson, andFoote.

It will be appreciated by those of ordinary skill in the art thatconventional or modified sonar techniques may be employed to locate apathogenic particle. For example, the acoustic radiation may bereflected back at the piezoelectric element 35, where the acousticenergy will be converted into electrical energy for analysis. Once theanalysis has been performed, a decision may be made as to whether and/orhow to dispense fluid from the reservoir. If no particle is sufficientlyclose to the surface for ejection, the acoustic energy may be focused atprogressively greater distances from the fluid surface until apathogenic particle is located and driven closer to the surface byfocused acoustic energy or other means. Similarly, the optimum intensityand directionality of the ejection acoustic wave may be determined fromsimilar types of acoustic analysis, optionally in combination withadditional data. For example, the desired intensity and directionalityof the ejection acoustic wave may be determined by using the data fromthe above-described assessment relating to reservoir volume or fluidproperty data, as well as geometric data associated with the reservoir.In addition, the data may show the need to reposition the acousticdevice so as to reposition the acoustic generator and/or focusing meanswith respect to the fluid surface, in order to ensure that the focalpoint of the ejection acoustic wave is near the fluid surface wheredesired. Thus, positioning means 36 may be used to ensure that theacoustic device 29 and reservoir 13 are appropriately positioned tocarry out acoustic ejection/detection with proper focus.

Thus, one advantage of the invention is the ability to selectivelydispense components of the pathogenic fluid. For example, one couldselect a droplet size too small to entrain a host cell and enableseparation of non-cell containing liquid from the pathogenic fluid.There are other methods to accomplish this type of separation includingdetecting the presence or absence of cells in the ejection zone beforeopting to dispense the droplet. Such sorting/selective dispensationfunctionalities are known in the art See, e.g., U.S. Patent ApplicationPublication No. 20020064808 to Mutz et al.; U.S. patent application Ser.No. 09/999,166, Publication No. 20020142286, filed Nov. 29, 2001, for“Focused Acoustic Energy for Ejection Cells from a Fluid,” inventorsMutz and Ellson, assigned to Picoliter Inc. (Mountain View, Calif.);U.S. Patent Application Publication No. 20020064809 to Mutz et al.; andU.S. patent application Ser. No. 10/040,926, Publication No.20020090720, filed Dec. 28, 2001, for “Focused Acoustic Ejection CellSorting System and Method,” inventors Mutz, Ellson, and Lee, assigned toPicoliter, Inc. (Mountain View Calif.).

In order to deposit droplets of fluid from the reservoir into the well51, a positioning means 52 is employed to align well 51 with reservoir13. The cover 57, as depicted in FIG. 1B, is removed from the well 51,and the well 51 is positioned above the reservoir 13 by the positioningmeans 52 such that the designated site 53 located on the interiorsurface of the substrate faces the surface 17 of the fluid 15 in thereservoir. Also as shown in FIG. 1B, culturing fluid 59 may beconstrained within the well 51 through surface forces. Once a particlethat is sufficiently close to the fluid surface 17 of reservoir 13 islocated and is determined to meet any other criteria for ejection, theacoustic device 29 serves as an ejector. The acoustic generator 31 isactivated to produce an acoustic wave that is focused by the focusingmeans 33 to eject a volume of fluid that forms droplet 61, whichcontains a pathogenic particle 19. Generally, an ejected droplet may notcontain more than one particle when the droplet to particle volume ratiois less than about 2:1. In some instances, however, a droplet maycontain a plurality of pathogenic particles. For example, a single viralparticle may have a cross-sectional dimension of about 10 nm. A 1 pLdroplet may contain a plurality of viral particles of this size. One wayin which the precise amount of energy required to eject only therequired volume and no more can be determined by slowly increasing theenergy applied, from an amount insufficient to eject a particle desiredfor ejection, until there is just enough energy applied to eject thedroplet the desired distance to the targeted substrate locale. Afterthis initial determination, approximately the same energy, withadjustment for any change in fluid level, may be applied to ejectparticles of substantially the same volume as the initial calibrationparticle. As a result, droplet 61 may be ejected from fluid surface 17into the interior of well 51. As depicted in FIG. 1B, the droplet 61 maycontain a single pathogenic particle 19. However, a plurality ofpathogenic may be ejected under some instances. In either case, theparticle or particles are thus exposed to the candidate compound inculturing fluid 59.

Then, as shown in FIG. 1C, the cover 57 is placed over the well 51 toform an enclosure that contains the ejected pathogenic particle 19suspended in the culturing fluid 59 with the candidate compound.Typically, the enclosure is sealed such that no matter enters or exitsfrom the enclosure while sealed. The enclosure may then be acousticallycoupled to the acoustic device so as to allow acoustic monitoring forchanges in the amount and/or concentration of the pathogen enclosed inthe enclosure. As depicted, the acoustic device 29 may be positionedbelow the well 51, in order to achieve acoustic coupling between theacoustic device and the well through the acoustic coupling medium 41.Once the acoustic device and the well are in proper alignment, thetransducer 35 of the acoustic generator 31 may be activated to produceacoustic radiation in order to perform an initial assessment of thecontents of the well. The assessment may be carried out in a similarmanner as set forth herein and in U.S. patent application Ser. No.10/010,972, Publication No. 20030101819, entitled “Acoustic Assessmentof Fluids in a Plurality of Reservoirs,” filed Dec. 4, 2001, byinventors Mutz, Ellson, and Foote, and may establish a baseline to whichlater assessments may be compared. As depicted in FIG. 1D, heatingelements 63 within the system enclosure 11 are activated to bring thecontents within the well to culturing conditions and the number ofpathogenic particles 19 within well 51 are increased. The acousticassessment may then be carried out at predetermined intervals todetermine whether there are any changes in the amount and/orconcentration of the pathogen as a result of interaction with thecandidate compound. Other forms of assessment, such as optical density,may be used.

It should be apparent that in some embodiments, the invention relates tothe use of a pathogen-impermeable enclosure as well as to the ejectionof a droplet of pathogenic fluid from a reservoir to a substrate.Accordingly, the invention also provides a device for dispensing one ormore droplets of fluid. The device includes a reservoir adapted tocontain a fluid, an ejector for applying focused radiation to thereservoir in a manner effective to eject a droplet of fluid from thereservoir, and a means for positioning a substrate such that a substrateis positioned to receive a droplet of fluid from the reservoir. Apathogen-impermeable enclosure is also provided for isolating thereservoir and substrate therein.

Any of a number of different ejectors that apply focused radiation maybe used. For instance, the ejector may apply focused electromagneticradiation of appropriate wavelengths to eject droplets from thereservoir. Typically, the ejector is an acoustic ejector. Althoughordinary inkjet technologies involving piezoelectric elements may beemployed, it is preferred that the ejector be a nozzleless acousticdevice that employs an acoustic generator and a focusing means forfocusing the acoustic radiation generated thereby. Exemplary ejectorsare described above and in U.S. Patent Application Publication No.20020037579 to Ellson et al.

The device typically includes a detachable reservoir to providemodularity and interchangeability of components. In addition, thereservoir may be adapted for single use. However, integrated orpermanently attached reservoirs may be employed as well. For any ofthese reservoirs, a pathogen-impermeable cover may be provided forestablishing sealing contact therewith.

As alluded to above, the invention may be used for pathogen formattingor reformatting purposes. Thus, a plurality of reservoirs and/orsubstrates may be provided for use with the present invention. Thereservoirs and/or substrates are preferably substantially acousticallyindistinguishable from each other. In addition, the reservoirs may beprovided in a single-piece unit, such as when the reservoirs representwells of a well plate. Many well plates suitable for use with the deviceare commercially available and may contain, for example, 96, 384, 1536,or 3456 wells per well plate. Manufacturers of suitable well plates foruse in the employed device include Corning, Inc. (Corning, N.Y.) andGreiner America, Inc. (Lake Mary, Fla.). However, the availability ofsuch commercially produced well plates does not preclude the manufactureand use of custom-made well plates containing at least about 10,000wells, or as many as 100,000 to 500,000 wells, or more. To facilitatehandling of multiple reservoirs, it is preferred that the reservoirs besubstantially acoustically indistinguishable from one another. Inaddition, the reservoirs should be generally arranged in a pattern or anarray to provide each reservoir with individual systematicaddressability.

The reservoirs or substrates may come into contact with pathogenicfluids, nonpathogenic fluids, or a combination of pathogenic andnonpathogenic fluids. Furthermore, the material used in the constructionof reservoirs and substrates should be compatible with the fluids incontact therewith. Thus, if it is intended that the reservoirs orsubstrates contact an organic solvent such as acetonitrile, polymersthat dissolve or swell in acetonitrile would be unsuitable for use informing the reservoirs or substrates. Similarly, reservoirs orsubstrates intended to contact DMSO should be compatible with DMSO.Additional information relating to materials selection and constructionof reservoirs and substrates, and to fluids with which the reservoirsand substrate may come into contact, are described, for example, in U.S.Patent Application Publication Nos. 20020037579 and 20020037375, each toEllson et al., and in U.S. Pat. Nos. 5,520,715 and 5,722,479 toOeftering.

In some instances, a microfluidic device may be used with the inventionin place of the substrate and/or reservoir. Microfluidic devicestypically have fluid-transporting features of micrometer orsubmicrometer dimensions in which any number of processes and oranalytical techniques involving very small amounts of fluid may becarried out. Microfluidic devices are available from ACLARA BioSciences,Inc. (Mountain View, Calif.), Caliper Technologies Corp. (Mountain View,Calif.), and Fluidigm Corp. (South San Francisco, Calif.). The combinedemployment of focused acoustic ejection and microfluidic devices isdiscussed in greater detail in U.S. patent application Ser. No.10/066,546, Publication No. 20020125424, entitled “Acoustic SampleIntroduction for Analysis and/or Processing,” filed Jan. 30, 2002, byinventors Ellson and Mutz.

Similarly, the device may further include a means for positioning theacoustic ejector as well as a means for positioning the substrate, suchas described, for example, in U.S. Patent Application Publication Nos.20020037579 and 20020037375, each to Ellson et al. As described in thesepublications, either or both positioning means may be constructed from,for example, motors, levers, pulleys, gears, a combination thereof, orother electromechanical or mechanical means known to one of ordinaryskill in the art. Furthermore, it will be appreciated that variouscomponents of the device may require individual control orsynchronization. Such control or synchronization may allow for theejection droplets to prepare an array on a substrate surface.

Furthermore, the device may also include certain performance-enhancingfeatures. For example, the device may include a cooling means forlowering the temperature of the substrate to ensure, for example, thatthe ejected droplets adhere to the substrate. The cooling means may beadapted to maintain the substrate surface at a temperature that allowsfluid to partially, or preferably substantially, solidify after thefluid comes into contact therewith. The device may also include a meansfor maintaining fluid in the reservoirs at an appropriate temperature,since repeated application of acoustic energy to a fluid will result inheating, which can in turn cause unwanted changes in fluid propertiessuch as viscosity, surface tension, and density. Design and constructionof such temperature-maintaining means are known to one of ordinary skillin the art and will involve incorporation of at least one heatingelement and/or at least one cooling element.

In addition, the device may further include a means for manuallymanipulating items within the enclosure without compromising thepathogenic impermeability of the enclosure. In addition, a locatingmeans may be provided for locating a discrete particle in the pathogenicfluid. Exemplary locating means are described in U.S. patent applicationSer. Nos. 09/727,391, 09/999,166, and 10/033,739 (Publication Nos.20020064808, 20020142286, and 20020090720 respectively). When the deviceincludes an acoustic generator, the locating means may include ananalyzer for analyzing acoustic radiation generated by the acousticgenerator. Such an analyzer is typically positioned to receive acousticradiation generated by the acoustic generator and transmitted throughfluid contained in the reservoir. In some instances, as described above,the analyzer is positioned to receive acoustic radiation reflected by afree fluid surface contained in the reservoir. In such a case, theanalyzer may include a component common to the acoustic generator, suchas a piezoelectric element. Exemplary analyzers are described in U.S.patent application Ser. No. 10/010,972, Publication No. 20030101819,entitled “Acoustic Assessment of Fluids in a Plurality of Reservoirs,”filed Dec. 4, 2001, by inventors Mutz, Ellson, and Foote.

It should be noted that acoustic assessment of the pathogenic contentsof a pathogen-impermeable enclosure may be performed in transmissivemode rather than in reflective mode. As depicted in FIG. 2, a device 22may be provided that includes a reservoir 13 adapted to contain a fluid14. As depicted, the reservoir 13 is in a sealed state. An acousticradiation generator 35 is positioned below the reservoirs, and analyzer38 is positioned in opposing relationship with the acoustic radiationgenerator 35 above the reservoirs. That is, the contents 14 of thereservoir 13 may undergo acoustic assessment when the reservoir 13 isinterposed between the acoustic radiation generator 35 and the analyzer38. The acoustic radiation generator 35 and the analyzer 38 areacoustically coupled to the reservoir via coupling media 41 and 42,respectively. Once the acoustic radiation generator 35, the reservoir13, and the analyzer 38 are in proper alignment, the acoustic radiationgenerator 35 is activated to produce acoustic radiation that istransmitted through the reservoir 13 and its contents 14 toward theanalyzer 38. The received acoustic radiation is analyzed by analyzer 38.

Thus, the invention also provides a method of monitoring for a change inthe amount and/or concentration of a pathogen in a pathogenic fluid. Asbefore, a pathogen-impermeable enclosure is provided that encloses apathogenic fluid comprising a pathogen and a carrier fluid. The methodalso involves acoustically monitoring for a change in the amount and/orconcentration of the pathogen enclosed in the pathogen-impermeableenclosure.

It should be apparent, then, that the invention permits previouslyunrealized opportunities in pathogenic studies through the use offocused acoustic radiation. For example, acoustic drop ejection may beused to array and/or rearray infectious pathogenic agents. This may beaccomplished, for instance, by using ordinary culturing techniques togrow a stock of bacterial pathogen in a single reservoir that containsculturing media. In some instances, the culturing medium may be providedas a coating on a solid surface. For example, the bacterial pathogen maybe immobilized on a substrate surface selected for facile immobilizationof cells. Such surfaces include, for example, a collagen-derivatizedsurface, dextran, polyacrylamide, nylon, polystyrene, and combinationsthereof. In some instances, the surfaces are inherently cytophilic. Inother instances, a cytophilic substrate surface is provided as a resultof surface modification. In a simple embodiment, the culturing mediummay be provided as a collagen coating on an interior surface of a well.

Focused radiation may then be used to transfer the culture to the wellsof a well plate, wherein each well contains a different antibioticcandidate compound. Then, each well plate may be sealed and put in anincubator for a bacterial growth assay. Every 30 minutes, an acoustictransducer could pass under each well to measure the reflected acousticenergy from a suitable acoustic pulse sent into the well and detecting achange due to the increase in the density of bacterial particles in theplate. In this way, growth rate inhibition for each compound could bedetermined.

As another example, drug compounds could be arrayed in a series ofconcentrations, allowing a dose response curve to be constructed.Throughout the process of candidate compound arraying, bacterialregrowth, and growth measurements, the assay plate would remain sealed.As a result, problems such as compound cross-contamination and thedanger of exposure of lab personnel to aerosolized pathogens aresubstantially reduced or eliminated. Since the acoustic frequencycontent of pulses sent in the wells may be varied, one could distinguishviral particles from bacterial, or bacterial cells from mammalian cells,in situ. Thus, acoustics could be used in the context of an assay wherean antibody library displayed on a phage would be used to assess theability of the antibody to protect mammalian cells from viral infection.The growth of mammalian cells could be readily be distinguished from thegrowth of bacteria or phage via acoustic detection, since mammaliancells are 10-1000 fold larger than bacterial or viral particles.

Furthermore, the assay system described herein can readily be expandedto the analysis of patient blood samples. Antibody and other tests canbe run in the sealed plate format, reducing the chance of exposure oflaboratory workers to aerosolized toxins, bacteria, or viruses, whileproviding a controlled environment for the performance of high-precisiondiagnostics. In addition, health workers could collect sample specimensand transfer them to microfluidic chips through the use of focusedradiation in sealed systems, for use in a laboratory or in fieldwork.

As another example, the invention may be used with a well platecomprised of a plurality of wells each containing a serum sample and acover or lid that has a substantially planar surface coated with afluorophore-conjugated antibody. The lid may be then sealed against thewell plate. After ejecting droplets from the wells to the coated lid, ahomogeneous fluorescence assay may be performed to detect the pathogenwhich has affinity to the antibody. Since the cover is sealed againstthe well plate, there is a lowered risk of pathogenic contamination. Asa result, the invention provides an improved method for carrying outsuch assays by reducing the risk of pathogenic exposure.

In some instance, the cover or lid may be patterned with a plurality ofdifferent conjugated antibodies bound thereto. Each region of the coveror lid may then be sealed over a well. The antibodies provide multiplefluorescent indicators for droplets of fluid ejected from the well.Based on the spatial position and other aspects of the fluorescentsignal, the antibody detection event can be identified.

This flat cover approach provides added advantages when used inconjunction with the invention. For example, it is a relatively simplematter to coat antibodies onto a flat surface. In addition, by using theflat cover, the depth of field for the optics in a fluorescent scanneris reduced. Decoupling the fluorescent read from the well plate is alsoadvantageous. It eliminates the background fluorescence of any materialsin the well and issues related to the opacity of the well plate itselfto the fluorescent wavelength used for detection. Also, if any wellcoating is required to promote an environment for amplifying thepathogen, the coating may be applied independently from the applicationof antibody coating on the cover.

Another advantage of the present invention is that it may be used inapplications wherein nucleotidic materials associated with pathogen areemployed and/or analyzed. For example, in order to determine whether anindividual has been infected with a viral or bacterial pathogen, bodilyfluid from the individual may be extracted to determine whethernucleotidic sequences associated with the pathogen is present in thebodily fluid. As such sequences may be present in a small amount,polymerase chain reaction (PCR) may be used in conjunction with theinvention.

PCR is a well known technique that makes it possible to start withotherwise undetectable amounts of nucleotidic material such as DNA andcreate ample amounts of the material for subsequent analysis. Inessence, PCR uses a repetitive series of steps to create copies ofpolynucleotide sequences located between two primer sequences. PCR firstinvolves mixing a template (e.g., target DNA to be amplified), twoprimer sequences selected so as to be complementary to a portion of thetemplate, PCR buffer, free deoxynucleotide tri-phosphates, such as dATP,dCTP, dGTP, and dTTP, and thermostable DNA polymerase. When a duplex DNAmolecule is used as the template, the DNA is denatured, using heat, intotwo complementary single strands. The primers then anneal to thestrands. A subsequent cooling step allows the primers to anneal tocomplementary sequences on single-stranded DNA molecules containing thesequence to be amplified. Replication of the target sequence is thenaccomplished by the DNA polymerase which produces a strand of DNA thatis complementary to the template. That is, nucleotide monophosphateresidues are linked to the primers in the presence of a thermostable DNApolymerase to create a primer extension product. After primer extension,twice as many duplex DNA molecules exist. Repetition of this processdoubles the number of copies of the sequence of interest, and multiplecycles increase the number of copies exponentially.

Since PCR requires repeated cycling between higher and lowertemperatures, PCR devices must be fabricated from materials capable ofwithstanding such temperature changes. In some instances, thermocyclingmay involve a denaturing step at around 90° C. to around 95° C. for 5 to60 seconds, an annealing step at around 50° C. to around 65° C. for 2 to80 seconds, and a polymerization step at around 72° C. for 5 to 120seconds. The sample may be subjected to 30 or more cycles to produce thedesired amplification. Thermocycling may be achieved by any suitable andconvenient method, e.g., using commercially available thermocyclers, aheating block apparatus, and/or an infrared radiation source inconjunction with cooling devices. After thermocycling is complete, a PCRsample may be cooled to a temperature of around 4° C. for subsequentanalysis, processing, treatment or testing. Thus, the materials fromwhich the pathogen-impermeable enclosures of the present invention aremade, including for example, any wells, lids, covers, etc. that serve ascomponents of the pathogen-impermeable enclosures, should bemechanically and chemically stable at high temperatures, and capable ofwithstanding repeated temperature changes without mechanicaldegradation. Furthermore, the materials should be compatible with thePCR reaction itself, and not inhibit the polymerase or bind DNA.Reactants for the PCR reaction may require encapsulation in awater-impermeable substance such as mineral oil to avoid drying in thethermocycling process.

It should be noted that other nucleic acid amplification and/or reactiontechniques are known in the art and that the term “PCR” encompasses suchadditional techniques as well. That is, the reference to the term “PCR”is intended to include ligase chain reactions, rolling circleamplification, repair chain reactions and other techniques involvingreaction mixtures that undergo denaturation, annealing and extensionprocesses.

The assay system as described above may be adapted for use with PCRtechniques. For example, in certain PCR based techniques, a sample maybe mixed with PCR reagents as well as with a detector for the presenceof DNA to quantify the DNA generated by the reaction and/or to determinethe presence or identity of a specific pathogen. Accordingly, anexemplary assay involves the ejection of pathogen-containing sampledroplets onto a lid, which may then be placed in sealing contact with awell of a well plate to form a pathogen-impermeable enclosure. Theejection may take place either before or after the sample is combinedwith the PCR reagents. Then, the enclosure is placed in a thermocyclerfor DNA amplification and for subsequent pathogen identification. Theresults of the assay can be determined by a variety of methods includingthe use of the optional DNA quantification material added to the PCRreagent. Methods for selection of primer pairs, both as positive andnegative controls for accurate pathogen identification and determinationof assay results, are known to those of skill in the art.

It is to be understood that while the invention has been described inconjunction with the preferred specific embodiments thereof, theforegoing description is intended to illustrate and not limit the scopeof the invention. Other aspects, advantages, and modifications will beapparent to those skilled in the art to which the invention pertains.

All patents, patent applications, journal articles, and other referencescited herein are incorporated by reference in their entireties.

We claim:
 1. A method for monitoring for a change in an amount, in aconcentration, or in both the amount and the concentration of a pathogenin a pathogenic fluid, comprising: (a) acoustically transferring atleast one droplet of the pathogenic fluid to a substrate; (b)transferring a compound to the substrate; (c) sealing the pathogenicfluid and the compound within a pathogen-impermeable enclosure byplacing a cover in sealing contact with the substrate; (d) performing aninitial acoustic assessment of the pathogenic fluid sealed within thepathogen-impermeable enclosure, wherein performing the initial acousticassessment includes: subsequent to step (c), generating, by apiezoelectric acoustic transducer disposed outside of thepathogen-impermeable enclosure, acoustic radiation directed towards thepathogen-impermeable enclosure; reflecting the acoustic radiation by thepathogenic fluid; receiving, by the piezoelectric acoustic transducer,the acoustic radiation reflected by the pathogenic fluid; and analyzingthe received acoustic radiation; (e) subsequent to step (d), subjectingthe pathogen-impermeable enclosure to a process altering the amount, theconcentration, or both the amount and the concentration of the pathogensealed in the pathogen-impermeable enclosure; and (f) subsequent to step(e), acoustically monitoring for the change in the amount, in theconcentration, or in both the amount and the concentration of thepathogen sealed in the pathogen-impermeable enclosure; wherein theacoustically monitoring for the change in the amount, in theconcentration, or in both the amount and the concentration of thepathogen sealed in the pathogen-impermeable enclosure includes:subsequent to step (e), generating, by the piezoelectric acoustictransducer, acoustic radiation directed towards the pathogen-impermeableenclosure; reflecting the acoustic radiation by the pathogenic fluid;receiving, by the piezoelectric acoustic transducer, the acousticradiation reflected by the pathogenic fluid; analyzing the receivedacoustic radiation; and comparing the analyzing of step (d) to theanalyzing of step (f).
 2. The method of claim 1, wherein the change isan increase in pathogen content.
 3. The method of claim 1, wherein thechange is a decrease in pathogen content.
 4. The method of claim 1,wherein the process of step (e) comprises subjecting the enclosure to atemperature change.
 5. The method of claim 4, wherein the process ofstep (e) comprises raising the temperature of the enclosure.
 6. Themethod of claim 4, wherein the process of step (e) comprises subjectingthe enclosure to a temperature selected to facilitate an increase in theamount, in the concentration, or in both the amount and theconcentration of the pathogen in the enclosure.
 7. The method of claim4, wherein the process of step (e) comprises subjecting the enclosure toa temperature selected to facilitate a decrease in the amount, in theconcentration, or in both the amount and the concentration of thepathogen in the enclosure.
 8. The method of claim 2, wherein theincrease in pathogen content is responsive to multiplication of thepathogen.
 9. The method of claim 3, wherein the decrease in pathogencontent is responsive to interaction of the pathogen with the compound.10. The method of claim 1, wherein the pathogen-impermeable enclosure isformed of polymer.
 11. The method of claim 1, wherein the pathogenicfluid comprises a culturing medium.
 12. The method of claim 1, whereinthe compound is transferred using acoustic radiation.
 13. The method ofclaim 1, wherein the substrate comprises a well of a well plate.
 14. Themethod of claim 1, wherein the compound comprises a nutrient.
 15. Themethod of claim 1, wherein the compound comprises a candidate drug. 16.The method of claim 1, wherein the compound comprises an antibody. 17.The method of claim 1, wherein the pathogenic fluid comprises a bloodsample of a patient.
 18. The method of claim 1, wherein the compoundcomprises a drug.